Methods for the treatment of tinnitus induced by cochlear excitotoxicity

ABSTRACT

The invention relates to methods for the prevention and/or treatment of tinnitus induced by cochlear excitotoxicity. In these methods, a pharmaceutical composition comprising an NMDA receptor antagonist is administered to an individual in need of such treatment by appropriate devices and/or formulations for local administration to the inner ear. The tinnitus to be prevented and/or treated may be provoked by acoustic trauma, presbycusis, ischemia, anoxia, treatment with one or more ototoxic medications, sudden deafness, or other cochlear excitotoxic-inducing occurrence. The invention also relates to method for the identification of compounds effective in the treatment and prevention of tinnitus by a novel screening method incorporating an electrophysiological test method.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for the delivery of pharmaceuticalcompounds to the inner ear for the treatment of tinnitus induced bycochlear excitotoxicity. Specifically, this invention relates to thelocal administration of N-Methyl-D-Aspartate (NMDA) receptor antagoniststo the inner ear to suppress the NMDA receptor mediated aberrantactivity of the auditory nerve following acute, repeated or prolonged orchronic occurrences of cochlear excitotoxicity provoked by incidentssuch as acoustic trauma, presbycusis, ischemia, anoxia, treatment withone or more certain ototoxic medications or sudden deafness and thus,block tinnitus in the case of such incidents.

2. Description of Related Art

Tinnitus, the perception of sound without external acoustic stimulation,is a very common inner ear disorder. It is estimated that 8.6 millionAmericans, about 3 percent of the U.S. population, suffer from chronictinnitus (Centers for Disease Control and Prevention, Vital and HealthStatistics, Series 10, #200, October 1999). According to the AmericanSpeech-Language-Hearing Association (ASHA), a million or more personsfind that their tinnitus pre-, vents them from leading a normal life(0.3% of the population). European population studies estimate 7% to 14%of the population have talked with their physician about tinnitus, whilepotentially disabling tinnitus occurs in approximately 1% to 2.4% ofpeople (Vesterarger V., British Medical Journal 314 (7082): 728-731(1997)).

In spite of the high prevalence of tinnitus and its severe impact on thehealth and quality of life of people affected by it, there is no trulyeffective treatment available. Current therapy approaches include theavoidance of ototoxic medications, reduced consumption of alcohol,caffeine and nicotine, reduced stress, the use of background noises orwearable tinnitus maskers (some in combination with hearing aids),behavioral therapies such as hypnosis, cognitive therapy andbiofeedback, tinnitus retraining therapy (TRT), pharmacological andother complementary therapies.

Tinnitus is not a disease, but rather a symptom common to varioushearing disorders, just as pain accompanies many different illnesses. Itis most frequently associated with noise-Induced hearing loss,presbycusis and Ménière's Disease (Nicolas-Puel et al., InternationalTinnitus Journal 8 (1): 37-44 (2002)). Other, less frequent originsinclude exposure to ototoxic drugs (aminoglycoside antibiotics,high-dose loop diuretics, nonsteroidal anti-inflammatory drugs andcertain chemotherapeutic agents), reduced vascular flow (ischemia),autoimmune processes, infectious diseases, conductive hearing loss,otosclerosis, head trauma etc. In over 90% of cases, tinnitus isassociated with hearing loss of known origin, and well over 70%originate within the inner ear (Nicolas-Puel et al., InternationalTinnitus Journal 8 (1): 37-44 (2002)).

Over the past decade, major advances in the research of thephysiopathology of the inner ear resulted in the identification of thekey role of the inner hair cell synaptic complex in the development oftinnitus induced by cochlear excitotoxicity, one of the most frequenttriggers of tinnitus. Excitotoxicity, which was first described by Olneyet al., J. Neuropathol. Exp. Neurol. 31(3): 464-488 (1972), is generallycharacterized as an excessive synaptic release of glutamate, which isthe most important neurotransmitter in the Central Nervous System aswell as in the auditory system. It activates postsynaptic glutamatereceptors (ionotropic and metabotropic), which leads to depolarizationand neuronal excitation. However, if receptor activation becomesexcessive by an excessive release of glutamate as in the case ofexcitotoxicity, the target neurons are damaged and may eventually die(Puel J. L, Prog Neurobiol. 47(6): 449-76 (1995)).

Cochlear excitotoxicity is provoked either by exposure to excessivenoise such as in the case of acute or repeated acoustic trauma (whichleads to noise-induced hearing loss or presbycusis), sudden deafness oranoxia/ischemia (Pujol and Puel, Ann. NY Acad. Sci. 884: 249-254 (1999))or treatment with one or more certain ototoxic medications. The releaseof excessive amounts of glutamate is induced either by the excessivesound pressure entering the cochlea in case of acoustic trauma or thereduced blood flow to the glutamate regulating system in case ofanoxia/ischemia respectively sudden deafness. In all cases,excitotoxicity is characterized by a two-step mechanism: first, there isan acute swelling of the type I afferent dendrites mediated by theionotropic glutamate receptors, which leads to a disruption of thepostsynaptic structures and a loss of function. Within the next 5 days,synaptic repair (neo-synaptogenesis) is observed with a full or partialrecovery of cochlear potentials (Puel et al., Acta Otolaryngol. 117 (2):214-218 (1997)). In the second phase of excitotoxicity, which maydevelop after strong and/or repetitive injury, a cascade of metabolicevents triggered by the entry of Ca²⁺ leads to neuronal death of thespiral ganglion neurons.

Cochlear excitotoxicity may induce tinnitus during the process ofrupturing of the postsynaptic structures and, provided the rupture isnot terminal, the following neo-synaptogenesis at the inner hair cellsynaptic complex (Puel et al., Audiol. Neurootol. 7 (1): 49-54 (2002)).A key role in functional recovery after excitotoxicity is played by theNMDA receptors, which are not involved in the activity of auditory nervefibres under physiological conditions (Puel et al., Audiol. Neurootol. 7(1): 49-54 (2002)), but are up-regulated during the process ofneo-synaptogenesis (Puel et al, C. R. Acad. Sci. III. 318 (1): 67-75(1995)), mainly owing to their high calcium (Ca²⁺) permeability (Sattlerand Tymianski, Mol. Neurobiol. 24 (1-3): 107-129 (2001)). As could beshown in an animal model of cochlear synaptic repair mechanisms,blockage of the NMDA receptors by local administration of the NMDAreceptor antagonist D-AP5 delayed the functional recovery and theregrowth of auditory dendrites (Gervais D'Aldin et al., Int. J. Dev.Neurosci. 15 (4-5); 619-629 (1997)). It could thus be concluded thatglutamate, in addition to its role as a fast excitatoryneurotransmitter, has a neurotrophic role via the activation of NMDAreceptors.

It has been hypothesized that the up-regulation of mRNA of NMDAreceptors induced by cochlear excitotoxicity is responsible for abnormalspontaneous “firing” of the auditory nerve fibres, which may beperceived as tinnitus (Puel J.-L. et al., Audiol. Neurootol, 7 (1):49-54 (2002)). During the process of neo-synaptogenesis afferentdendrites are in a critical state, and may thus be particularlysusceptible to excitation by the activation of the NMDA receptors. Toavoid any such aberrant excitation, and therefore tinnitus, which maywell continue infinitely due to incomplete neo-synaptogenesis, atherapeutic strategy would thus seek to specifically antagonize NMDAreceptors. As has been demonstrated, the local administration of NMDAreceptor antagonists to the cochlea prevents excitotoxicity induced byacoustic trauma or ischemia (Duan et al., Proc. Natl. Acad. Sci. USA 97(13): 7597-7602 (2000); Puel, Prog. Neurobiol. 47 (6): 449-476 (1995);Puel et al., J. Comp. Neurol. 341 (2): 241-256 (1994)). Whileexcitotoxicity could also be blocked by application of2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionate (AMPA) or kainatereceptor antagonists, as the acute swelling of afferent dendritesprimarily occurs via them (Puel et al., J. Comp. Neurol. 341 (2):241-256 (1994)), such an approach would have a potentially significantlynegative impact on the auditory function. As fast excitatoryneurotransmission between the inner hair cells and the auditory nervefibres is predominantly mediated by AMPA preferring receptors (Ruel etal., J. Physiol. London 518: 667-680 (1999)), their blocking wouldsuppress not only the undesired excessive stimulation of the auditorynerve, but also the desired, regular excitation and thus provoke hearingloss.

The hypothesized implication of NMDA receptors in the generation oftinnitus has so far only been tested and demonstrated in vivo with abehavioral model of salicylate-induced tinnitus (Guitton et al., J. ofNeuroscience 23 (9): 3944-3952 (2003)). The behavioral model, which hadto be developed to measure tinnitus, as tinnitus is not directlyobservable, was based on the active avoidance paradigm: the animals wereconditioned to jump onto a pole whenever hearing a particular sound.Administration of salicylate led to a significant increase in the numberof jumps even in the absence of external sound (false positives),indicating the perception of tinnitus. Following delivery of the NMDAantagonists MK-801, 7-CK and gacyclidine to the animals' cochleas viathe round window membrane the number of false positives decreasedsignificantly, indicating the suppression of tinnitus.

While these results provided for the first time a confirmation of thehypothesized implication of NMDA receptors in the occurrence oftinnitus, they could clearly not be generalized for all kinds of thisinner ear disorder, as salicylate-induced tinnitus is a very peculiarform of tinnitus. Salicylate, the active component of aspirin, has beenknown for more than a century to induce tinnitus if taken in large doses(Cazals Y., Prog. Neurobiol. 62: 583-631 (2000)). It may provoke similarsensations of tinnitus as in the case of cochlear excitotoxicity orother cases with different origin, but it is usually reversible andbased on a specific molecular mechanism. Application of mefenamate, awell known cyclooxygenase inhibitor, instead of salicylate alsoincreased the number of false positive responses, suggesting thatsalicylate-induced tinnitus is related to an inhibition ofcyclooxygenase pathway. While tinnitus induced by cochlearexcitotoxicity is the result of a cascade of glutamate mediatedprocesses leading to the up-regulation of mRNA of NMDA receptors,salicylate-induced tinnitus is mediated by changes in the arachidonicacid metabolism (see e.g. Cazals Y., Prog. Neurobiol. 62: 583-631(2000)). Salicylate has been shown to inhibit cyclooxygenase activity(see e.g. Vane and Botting, Am. J. Med. 104: 2S-8S (1998)). Evidencedemonstrates that arachidonic acid potentiates NMDA receptor currents(Miller et al., Nature 355: 722-725 (1992); Horimoto et al., NeuroReport7: 2463-2467 (1996); Casado and Ascher, J. Physiol. 513: 317-330(1998)). Electrophysiological studies have demonstrated that arachidonicacid increases the channel opening probability of NMDA receptor invarious systems, including cerebellar granule cells, dissociatedpyramidal cells, cortical neurons, and adult hippocampal slices (seee.g. Miller et al., Nature 355: 722-725 (1992); Horimoto et al.,NeuroReport 7: 2463-2467 (1996); Yamakura and Shimoji, Prog. Neurobiol.59: 279-298 (1999)). Unlike tinnitus induced by excitotoxicity, there isthus no morphological damage to the inner hair cell synaptic complex,and in particular to the synaptic ending, involved in salicylate-inducedtinnitus.

U.S. Pat. No. 5,716,961 to Sands (incorporated herein by reference)discloses the administration of an NMDA receptor-specific antagonist forthe purpose of treating tinnitus. Its neuroprotective properties in thecase of glutamate excitotoxicity are demonstrated in cell culture.However, the compound's pharmacological action and efficacy underpathophysiological conditions in vivo are not shown, i.e. there is norelation to tinnitus induced by cochlear excitotoxicity. This must beconsidered a serious deficiency given the complexities of the inner haircell synaptic complex. In addition, Sands teaches oral administration ofthe NMDA receptor antagonist, while discussing topical administrationonly for cases where a patient is unable to swallow or the oral route ofadministration is otherwise impaired. Topical administration isdiscussed nonspecifically in the form of “solutions, lotions, ointments,salves and the like.”

Systemic administration of NMDA receptor antagonists to treat inner eardisorders is usually ineffective, as the cochlea is protected like thebrain by a biological barrier. Relatively high doses to achieve adesired therapeutic effect would thus be required, but various potentside effects of NMDA receptor antagonists such as reduced learning,memory or motility significantly restrict the maximum tolerable doses.As various studies with humans for the treatment of CNS disorders byNMDA receptor antagonists have shown, plasma levels after systemicadministration were consistently below those needed for maximalneuroprotection in animal models, as clinical doses had to be limiteddue to a number of potentially adverse CNS effects, catatonia, increasedblood pressure and anaesthesia (Kemp and McKernan, Nature Neuroscience5, supplement: 1039-1042 (2002)). On the other hand, it has been shownthat local administration of the NMDA-AMPA receptor antagonistcaroverine to the inner ear results in higher intra-cochlearconcentrations, while avoiding high secondary concentrations in plasmaand cerebrospinal fluid as seen with systemic administration (Chen etal., Audiol. Neurootol. 8: 49-56 (2003)).

U.S. Pat. No. 6,066,652 to Zenner et al. (incorporated herein byreference) discloses a method for treating tinnitus throughadministration of adamantane, a known NMDA receptor antagonist. Theinventors cite results from a clinical study with systemicadministration which showed a reduction in tinnitus during treatment.Hypotheses brought forward to explain the results obtained centre onouter hair cells and the presynapse, and do not specifically cover therole of NMDA receptors.

While there are several indications supporting the hypothesis that NMDAreceptors play an important role in the genesis of tinnitus induced bycochlear excitotoxicity, the foregoing discussion shows that themolecular mechanisms are still unclear, and that it is therefore notpossible to predict reliably whether the use of NMDA receptorantagonists will effectively block this particular type of tinnitus.Further pathophysiological studies on the generation of tinnitus arethus required to validate the hypothesis and develop specific and trulyeffective therapeutic strategies.

SUMMARY OF THE INVENTION

The invention relates to methods for preventing and/or treating tinnitusinduced by cochlear excitotoxicity in a human. The methods includeadministering to a human a therapeutically effective amount of apharmaceutical composition comprising an NMDA receptor antagonist. In amethod for treating tinnitus, the NMDA receptor antagonist administeredis effective to suppress or reduce NMDA receptor mediated aberrantactivity of the auditory nerve in the human in need of such treatment.In a method for preventing tinnitus, the NMDA receptor antagonistadministered is effective to prevent NMDA receptor mediated aberrantactivity of the auditory nerve in the human in need of such treatment.The tinnitus to be prevented and/or treated may be provoked by acoustictrauma, presbycusis, ischemia, anoxia, treatment with one or moreototoxic medications, sudden deafness, or other cochlearexcitotoxic-inducing occurrence.

The present invention also relates to novel methods for the screening ofcompounds for the treatment and prevention of tinnitus wherein themethod utilizes an electrophysiological method of measuring andquantifying the extent of tinnitus. The methods include theadministration to a test animal of a test compound wherein the testanimal, for example, comprises an electrode in contact with the roundwindow membrane of the ear. The electrode is used to measure theensemble spontaneous activity (ESA) of the ear where a spectral peak atabout 200 to 250 Hz is indicative of tinnitus. The administration oftest compounds to the animal, to the round window membrane or to theinner ear is embodied in the present invention. The animal may haveacquired tinnitus by, for example, acoustic trauma, presbycusis,ischemia, anoxia, treatment with one or more ototoxic medications,sudden deafness, or other equivalent cochlear excitotoxic-inducingoccurrence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows CAP measurements 7 days after trauma in control animals.Hearing loss induced by acoustic trauma was assessed by recording CAPmeasurements 7 days after trauma. A permanent threshold shift ofmaximally 13 dB±2.0 was observed at 10 kHz on the seventh day followingacoustic trauma.

FIG. 2 illustrates the measurement of score and false positive responsesfollowing acoustic trauma in control animals. (A) Acoustic trauma led toa decrease in correct behavioral responses to sound stimulation,followed by partial recovery over time, reflecting the induced hearingloss. (B) The number of false positives differed substantially amongtested animals following acoustic trauma. Group 1 animals did notexperience tinnitus, group 2 animals experienced tinnitus onlytransiently, while group 3 animals experienced tinnitus both transientlyand then permanently.

FIG. 3 illustrates that hair cell loss resp. hearing loss induced byacoustic trauma does not play a significant role in the generation oftinnitus. Treatment with D-JNKI-1 prevented hearing loss after acuteacoustic trauma, as shown by the rapid recovery of score followingtrauma (A) but had no significant effect on the prevention of tinnitus,as the prevalence and patterns of tinnitus were essentially the same asin untreated animals (B).

FIG. 4, illustrates that local administration of the NMDA antagonist7-CK to the round window membrane resulted in the prevention oftinnitus. (A) The average behavioral score dropped from day 0 to day 1and recovered subsequently; however improvement was slower than inuntreated animals. (B) Local administration of the NMDA antagonist 7-CKresulted in suppressing persistent tinnitus induced by cochlearexcitotoxicity; only cases of transient tinnitus could be observed.

FIG. 5 illustrates that local administration of the NMDA antagonistS-(+)-Ketamine to the round window membrane resulted in the preventionof persistent tinnitus. (A) The average behavioral score dropped fromday 0 to day 1 and recovered subsequently; however improvement wasslower than in untreated animals. (B) Local administration of the NMDAantagonist S(+)-Ketamine resulted in suppressing persistent tinnitusinduced by cochlear excitotoxicity, only cases of transient tinnituscould be observed.

FIG. 6 illustrates that acoustic trauma produces substantialmorphological damage on the sensory outer hair cells (OHC) and innerhair cell (IHC) synaptic complex, whereas administration of salicylatedoes not. The stereocilia of OHC and IHC remain intact followingsalicylate injections over 2 days (6A), while those animals which wereexposed to acoustic trauma display severe damage to OHC stereociliarybundles as well as disarrayed and in some cases even fused (indicated byblack arrowhead) IHC stereocilia (6D). Low magnification of the IHCsynaptic complex shows no ultrastructural abnormalities in case of thesalicylate treatment (6B), whereas in traumatized animals a massive anddramatic swelling (indicated by asterisks) of the radial afferentdendrites can be seen at the basal pole of the IHC in the affectedfrequency range area, confirming that excitotoxicity has occurred (6E).Note the presence of numerous vacuoles in the apical part of the IHC andabnormally shaped stereocilia (indicated by arrowhead). Highmagnification of the basal IHC pole shows again no anomalies for thesalicylate treated animal (6C). The two afferent nerve endings(indicated by a1 and a2) are normal, and a characteristic presynapticbody facing afferent a2 is clearly visible. Contrary to this, (6F)displays swollen (a1) and disrupted (a2) nerve endings and thepresynaptic body facing a2. Scale bars for A and D are 10 μm (scanningelectron microscopy), for B and E 5 μm, and for C and F 0.25 μm (alltransmission electron microscopy).

FIG. 7 illustrates the expression of the NR1 subunit of the cochlearNMDA receptor following exposure to salicylate or acoustic trauma,determined by Western Blot immunodetection. As can be seen, thesalicylate treatment did not induce any significant modification of NR1NMDA receptor subunit expression (4% higher than control animals). Incontrast, acoustic trauma led to a clear overexpression 5 days after theincident (+50% over control animals), which is consistent with theobservation of persisting tinnitus. However, 24 hours post trauma, thereis no significant overexpression detectable (+8%), which suggests thatthe mechanisms of transitory and permanent tinnitus after acoustictrauma are fundamentally different. Immunoblots from the brain ofcontrol animals were performed to verify that the molecular weight ofthe NR1 subunit is identical in both brain and cochlea.

FIG. 8 shows the ensemble spontaneous activity (ESA) of the cochlearnerve in guinea pigs following cochlear excitotoxicity which inducestinnitus and the effects of a subsequent treatment by an NMDAantagonist. (A) shows the permanent threshold shift (PTS) of theauditory function resulting from the exposure to noise at the beginningof the experiment. (B) shows the ESA recording just prior to theexposure of the animal to 130 dB of sound at 6 kHz during 40 minutes.Note the spectral peak centered at around 900 Hz to 1 kHz which isindicative of a normal hearing activity. (C) shows the ESA recording 5days after the noise exposure on T0, Note the spectral peak in the 200to 250 frequency range, which is indicative of the presence of tinnitus.This peak had appeared immediately after the noise trauma and persistedfrom then on in a stable way. The regular spectral peak, however, haddisappeared and subsequently recovered. The contralateral ear showedexactly the same pattern of ESA with a peak at 200 to 250 Hz, indicatingalso the presence of tinnitus. Following these measurements, the animalwas treated on T0 with the NMDA antagonist Ketamine by localadministration onto the round window membrane. (D) On the day followingtreatment administration (T1), the ESA recording for the treated earshowed the absence of the 200 to 250 Hz spectral peak, indicating thesuccessful suppression of tinnitus. The regular peak centered at 900 Hzto 1 kHz still appeared, yet diminished. In the contralateral ear,tinnitus was still measured. (E) 10 days after treatment (T+10) thespectral peak at 200 to 250 Hz still remained absent, demonstrating alasting effect of the treatment. In addition, the regular peak centeredat around 900 Hz to 1 kHz had fully recovered. (F) In the untreated leftear, the presence of tinnitus was still indicated by the ESA recording.Please note that the PTS was not changed by the treatment (A), i.e.hearing remained unaffected, which indicates that the treatment is notonly effective, but also does not produce any side effects.

DETAILED DESCRIPTION OF THE INVENTION Overview

The present invention is based on experimental findings with an animalmodel of tinnitus induced by cochlear excitotoxicity. The inventionrelates to the use of pharmaceutical compounds that act specifically asNMDA receptor antagonists. While not wishing to be bound by theory, itis believed that an NMDA receptor antagonist of the present inventionbinds to the NMDA receptor at one of its various binding sites, therebyblocking (partly or entirely) the opening of the receptor's ion channel.The NMDA receptor is activated in a complex manner such that bothglutamate and glycine binding are required to open the ion channel andpermit calcium entry (Kemp and McKernan, Nature Neuroscience 5,supplement: 1039-1042 (2002)). Glutamate has the neurotransmitter role,as it is released from presynaptic terminals in an activity-dependentmanner, whereas glycine acts as a modulator, which is present in theextracellular fluid at more constant levels. The ion-channel integral tothe NMDA receptor is voltage-dependently blocked by magnesium, anddepolarization removes this block. Binding of an NMDA receptorantagonist to either of the three antagonist sites results in partial orcomplete blockage of the NMDA receptor and hence blocks or reduces theopening of the ion channel and depolarization of the neuron. The NMDAreceptor antagonist thus suppresses the aberrant excitation of theauditory nerve through up-regulated NMDA receptors which may followcochlear excitotoxicity and thus also reduces or eliminates theperception of tinnitus. Following delivery of the NMDA receptorantagonist, the NMDA receptors are no longer up-regulated. By targetingspecifically NMDA receptors, which are only up-regulated underpathophysiological conditions, to suppress the NMDA receptor mediatedaberrant activity of the auditory nerve, undesired side-effects onhearing can be avoided, as normal auditory neurotransmission isprimarily mediated by AMPA receptors.

In one embodiment, the invention relates to a method for treatingtinnitus induced by cochlear excitotoxicity in a human. The methodcomprises administering to a human a therapeutically effective amount ofa pharmaceutical composition comprising an NMDA receptor antagonist. TheNMDA receptor antagonist is administered in an amount and for a periodof time, effective to suppress or reduce NMDA receptor-mediated aberrantactivity of the auditory nerve in a human in heed of such treatment.Suppression or reduction of the NMDA receptor-mediated aberrant activityof the auditory nerve results in suppression or reduction of thetinnitus in the treated individual. In a preferred embodiment of thismethod, the NMDA receptor antagonist is administered after or during thehuman's exposure to a cochlear excitotoxic-inducing occurrence.

In another embodiment, the invention relates to a method for preventingtinnitus induced by cochlear excitotoxicity in a human. This methodcomprises administering to a human a therapeutically effective amount ofa pharmaceutical composition comprising an NMDA receptor antagonist. Inthis method the NMDA receptor antagonist is administered in an amountand for a period of time, effective to prevent NMDA receptor-mediatedaberrant activity of the auditory nerve in an individual in need of suchtreatment. Prevention of NMDA receptor-mediated aberrant activity of theauditory nerve prevents tinnitus in the treated individual. In apreferred embodiment of this method, the NMDA receptor antagonist isadministered prior to or during the human's exposure to a potentialcochlear excitotoxic-inducing occurrence. It is an object of the presentinvention to prevent and/or treat tinnitus which has been induced bycochlear excitotoxicity. It is not a requirement that the tinnitusinduced by cochlear excitotoxicity be provoked by any specific type ofoccurrence, only that the occurrence provoke cochlear excitoxicity andinduce tinnitus. It is not necessarily a requirement that the nature ofthe occurrence be known in preventing and/or treating tinnitus. Thetinnitus prevented and/or treated may be acute, subacute, or chronic.

In another embodiment, the present invention relates to methods ofscreening compounds for the treatment and/or prevention of tinnitus. Themethods of the present invention comprise the administration of acompound to an animal that has tinnitus as measured by an ensemblespontaneous activity (ESA) with a spectral peak centered at about 200 to250 Hz. Such conditions may be caused by, for example, acoustic traumasuch as, for example, cochlear excitotoxicity. The compound may beadministered, for example, locally, to the cochlear round windowmembrane or directly into the inner ear. The reduction in the ensemblespontaneous activity (ESA) with a spectral peak centered at about 200 to250 Hz to a lower value is indicative of a reduction in the level oftinnitus experienced by the animal. The level in the reduction of theESA can be compared to either, for example, a control animal sufferingfrom tinnitus treated with a control agent, the untreated (or controlagent treated) ear on the test animal or historic data. Although themethods of the present invention can be used to screen any compound forthe ability to treat tinnitus, in a preferred embodiment, the compoundis an NMDA receptor antagonist. The ESA measurement is obtained, forexample, through an electrode in contact with the round window membraneof the ear and is read in relationship to a second electrode located inanother part of the body such as, for example, neck muscle. It isanother aspect of the present invention that tinnitus may be induced,for example, by exposing the animal to acoustic trauma, an example ofwhich is subjecting the animal to a noise of about 130 dB at about 6 kHzfor about 40 minutes.

In another embodiment, the present invention also relates to anelectrophysical method for identifying compounds effective in theprevention of Tinnitus. In one embodiment, the method comprisesadministering a test compound to a test animal and exposing both thetest animal and a control animal to conditions capable of inducingtinnitus as determined by measuring the ESA to determine if the spectralpeak at about 200 to 250 Hz, wherein the reduction of the spectral peakat about 200 to 250 Hz in the test animal as compared to the controlanimal is indicative of the prevention of tinnitus. Although the methodsof the present invention can be used to screen any compound for theability to prevent tinnitus, in a preferred embodiment, the compound isan NMDA receptor antagonist. The ESA measurement is obtained, forexample, through an electrode in contact with the round window membraneof the ear and is read in relationship to a second electrode located inanother part of the body such as, for example, neck muscle.

It is known in the art that tinnitus results from cochlearexcitotoxicity following acoustic trauma, prebycusis, ischemia, anoxia,treatment with one or more certain ototoxic medications and/or suddendeafness. The prevention of tinnitus induced by acoustic trauma isexemplified herein, One of skill in the art would predict with a highdegree of certainty that the methods provided herein would be effectivein preventing and/or treating tinnitus induced not only by acoustictrauma, but also by prebycusis, ischemia, anoxia, treatment with one ormore certain ototoxic medications and/or sudden deafness since tinnitusresulting from all such occurrences share a common mechanistic cause.The acoustic trauma, prebycusis, ischemia, anoxia, treatment with one ormore certain ototoxic medications and/or sudden deafness may becharacterized as acute, repeated, or prolonged. One of skill in the artwould predict that the methods of the present invention would beeffective in preventing and/or treating tinnitus induced by means otherthan acoustic trauma, prebycusis, ischemia, anoxia, treatment with oneor more certain ototoxic medications and/or sudden deafness as long asthe tinnitus is induced by cochlear excitotoxicity. The cochlearexcitoxicity resulting from such occurrences may be characterized asacute, repeated, or prolonged, depending on the duration of the cochlearexcitotoxic-inducing occurrence.

The term “ototoxic medication,” as used in the context of the presentinvention, is intended to mean any compound characterized by the abilityto induce tinnitus via cochlear excitotoxicity upon therapeuticadministration. Cochlear excitotoxicity arises as a side-effect of theadministration of ototoxic medications, which are generally administeredas therapeutic compounds for treating conditions which may be unrelatedto hearing or hearing perception. Ototoxic medications characterized assuch include, for example, aminoglycoside antibiotics andchemotherapeutic agents such as cisplatin. Correlation of use of suchototoxic medications with cochlear excitotoxicity and the incidence oftinnitus is well known in the art, but prior to the present invention,no effective treatment has been available. The use of many suchmedications is currently limited by their ototoxic effects, and as sucha method for reducing these effects would enable such medications to beused more widely as therapeutics. “Ototoxic,” as used in the context ofthe present invention, is intended to mean any compound characterized byhaving a deleterious effect upon either the eighth nerve or upon theorgans of hearing and balance.

Compound

Formulations of the pharmaceutical compounds to be administered inconnection with the methods of the present invention comprise aselective NMDA receptor antagonist which binds to the NMDA receptoreither at the competitive NMDA antagonist binding site, thenon-competitive NMDA antagonist binding site within the on channel, orto the glycine site, Exemplary compounds include, but are notnecessarily limited to, ifenprodil, Ketamine, memantine, dizocilpine(MK-801), gacyclidine, traxoprodil (non-competitive NMDA antagonists),D-2-amino-5-phosphonopentanoic acid (D-AP5),3-((±)2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP),conantokins (competitive NMDA antagonists), 7-chiorokynurenate (7-CK),and Licostinel (glycine site antagonists). An NMDA antagonist for use inthe present invention may be any derivative, analogue, and/orenantiomeric form of an NMDA antagonist thereof which retains thefunction of an NMDA antagonist. The composition for administration inthe methods of the present invention may comprise one or more NMDAreceptor antagonists.

Ketamine, one of the preferred compounds of the present invention,belongs to the class of aryicycloalkylamines, and any derivative,analogue, and/or enantiomeric form of ketamine or arylcycloalkylaminethat retains the function of an NMDA antagonist may be used inconjunction with the present invention. Amongst the class ofarylcycloalkylamines compounds having the general formula I,

wherein R1, R2, R3, R4 and R5 independently are H, Cl, F, I, CH₃,CH₂CH₃, NH₂, OH or COOH and wherein R6 and R7 are independently H, CH₃,CH₂CH₃, OH, CI, F, or I may be preferred.

A preferred arylcycloalkylamine is ketamine (C₁₃H₁₆CINO (free base),2-(2-chlorophenyl)-2-(methylamino)-cyclohexanone), the structuralformula of which is represented by formula II.

Ketamine is a non-competitive NMDA-receptor antagonist which binds tothe PCP-binding site, a separate site of the NMDA-receptor complexlocated within the ion channel, thereby blocking the transmembranous ionflux. Ketamine may be provided by methods disclosed in U.S. Pat. No.3,254,124. More specifically, the preferred compound is (S)-Ketamine, asit binds with a 3-4-fold higher affinity to the PCP binding site of theNMDA receptor than (R)-ketamine (Vollenweider et al., Eur.Neuropsychopharmacol. 7: 25-38 (1997)). The synthesis of the opticalisomers may be carried out as described by DE 2062620 or WO01/98265,which are incorporated herein by reference. In a preferred embodiment ofthe present invention ketamine may also be administered as hydrochloridesalt (C₁₃H₁₇Cl₂NO) of its free base form (ketamine hydrochloride).

Another preferred compound, 7-chiorokynurenate (7-CK), is represented bythe following structure of formula III.

Any derivative or analogue of 7-CK may also be used in methods of thepresent invention.

Administration and Formulation

Delivery of the compound to patients can be accomplished orally,intravenously, subcutaneously, intraperitoneally, intramuscularly,rectally or topically, whereas topical administration to the inner earis generally preferred, as therapeutically effective doses with systemicadministration may induce undesired side-effects. One of skill in theart will recognize that administration of an NMDA antagonist in thepresent invention may be accomplished in a variety of other ways. Theonly requirement for administration in the present invention is that atherapeutically effective amount of a pharmaceutical compositioncomprising an NMDA antagonist be able to reach the site of the NMDAreceptor mediated aberrant activity of the auditory nerve in theafflicted individual.

Administration of the compound to the inner ear may be accomplished byvarious delivery techniques. These include the use of devices or drugcarriers to transport and/or deliver the compound in a targeted fashionto the membranes of the round or oval window, where it diffuses into theinner ear or is actively infused. Examples are otowicks (see e.g. U.S.Pat. No. 6,120,484 to Silverstein, incorporated herein by reference),round window catheters (see e.g. U.S. Pat. Nos. 5,421,818; 5,474,529;5,476,446; 6,045,528; all to Arenberg, or U.S. Pat. No. 6,377,849 andits division 2002/0082554 to Lenarz, all of which are incorporatedherein by reference), or various types of gels, foams, fibrins or otherdrug carriers, which are placed in the round window niche or on the ovalwindow, and loaded with the compound for sustained release (see e.g. WO97/38698 by Manning; Silverstein et al., Otolaryngology—Head and NeckSurgery 120 (5): 649-655 (1999); Balough et al., Otolaryngology—Head andNeck Surgery 119 (6): 427-431 (1998)). They further include the use ofdevices which are inserted into the cochlear duct or any other part ofthe cochlea (see e.g. U.S. Pat. No. 6,309,410 to Kuzma, incorporatedherein by reference). The compound may also be administered to the innerear by transtympanic injection, where the middle ear or part of it isfilled by a solution or other carriers of the compound (see e.g. Hofferet al., Otolaryngologic Clinics of North America 36 (2): 353-358(2003)). The preferred method of administration to the inner ear is bydiffusion across the round window membrane, which is relatively easilyaccessible from the middle ear space, and allows the inner ear to remainintact, thus avoiding any potential problems from leaking intracochlearfluids.

A compound contained within the pharmaceutical composition of thisinvention may be provided in the form of a pharmaceutically acceptablesalt. Examples of such a salt include, but are not limited to, thoseformed with organic acids (e.g. acetic, lactic, citric, malic, formaric,tartaric, stearic, ascorbic, succinic, benzoic, methanesulfonic,toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloridic,nitric, diphosphoric, sulphuric, or phosphoric acid), and polymericacids (e.g., tannic acid, carboxymethyl cellulose, polylactic,polyglycolic, or co-polymers of polylactic-glycolic acids).

Pharmaceutical compositions for any route of administration of thisinvention contain a therapeutically effective amount of activeingredient, and, as may be necessary, inorganic or organic, solid orliquid pharmaceutically acceptable carriers. Pharmaceutical compositionssuited for topical administration to the inner ear include aqueoussolutions or suspensions, which, e.g. In the case of lyophilizedformulations that contain the active ingredient alone or together with acarrier, may be prepared prior to use. They further include gels, whichmay be biodegradable or non-biodegradable, aqueous or non-aqueous, ormicrosphere based. Examples of such a gel include, but are not limitedto, poloxamers, hyaluronates, xyloglucans, chitosans, polyesters,poly(lactides), poly(glycolide) or their co-polymers PLGA, sucroseacetate isobutyrate, and glycerol monooleate. Pharmaceuticalcompositions suited for enteral or parenteral administration includetablets or gelatine capsules or aqueous solutions or suspensions asdescribed above.

The pharmaceutical compositions may be sterilized and/or may containadjuvants, e.g. preservatives, stabilizers, wetting agents and/oremulsifiers, salts for regulating the osmotic pressure and/or buffers.The pharmaceutical compositions of the invention may, if desired,contain further pharmacologically active substances, They may beprepared by any of the methods well known in the art of pharmacy, e.g.by conventional mixing, granulating, confectioning, dissolving orlyophilizing methods, and contain from about 0.01 to 100%, preferablyfrom about 0.1 to 50% (lyophilisates up to 100%), of active ingredient.

In a preferred embodiment the pharmaceutical composition according tothe invention is formulated for topical application. Suitable vehiclesfor otic administration are organic or inorganic substances, which arepharmaceutically acceptable and which do not react with the activecompounds, for example saline, alcohols, vegetable oils, benzylalcohols, alkylene glycols, polyethylene glycols, glycerol triacetate,gelatin, carbohydrates such as lactose or starch, magnesium, stearate,talc and petrolatum. The indicated preparations can be sterilized and/orcontain ancillary substances such as lubricants, preservatives, such asthiomersal (e.g., at 50%), stabilizers and/or wetting agents,emulsifiers, salts to influence the osmotic pressure, buffer substances,colorants, and/or aromatizing substances. They can, if required, alsocontain one or more other active ingredients. Otic compositions inaccordance with the present invention can comprise various ingredients,including other biologically-active-agents, such as antibiotics, e.g.,fluoroquinolones, anti-inflammatory agents, e.g., steroids, cortisone,analgesics, antipyrine, benzocaine, procaine, etc.

Compositions of the present invention for topical administration cancomprise other ingredients which are pharmaceutically-acceptable. Inpreferred embodiments of the present invention, a topical excipient isselected that does not enhance delivery of the agent to the systemiccirculation or to the central nervous system when administered to theear. For example, in general, it is preferred that the topical excipientnot have substantial occlusive properties, which enhance percutaneoustransmission through the mucosa into the systemic circulation. Suchocclusive vehicles include hydrocarbon bases, anhydrous absorption basessuch as hydrophilic petrolatum and anhydrous lanolin (e.g., Aquaphor),and water-in-oil emulsion bases such as lanolin and cold cream. Morepreferred are vehicles which are substantially non-occlusive and,generally, include those which are water-soluble, such as oil-in-wateremulsion bases (creams or hydrophilic ointments) and water-soluble basessuch as polyethylene glycol-based vehicles and aqueous solutions gelledwith various agents such as methylcellulose, hydroxyethyl cellulose andhydroxypropylmethylcellulose (e.g., K Y Gel).

Suitable topical excipients and vehicles can be routinely selected for aparticular use by those skilled in the art, and especially withreference to one of many standard texts in the art, such as Remington'sPharmaceutical Sciences, Vol. 18, Mack Publishing Co., Easton, Pa.(1990), in particular Chapter 87. For instance, biologically activeagents in accordance with the present invention can be combined withenhancing agents which enhance the penetration of an agent.

The compound can be administered prior to, during or after tinnitus hasbeen induced by excitotoxicity. The amount to be administered may vary,depending upon the method of administration, duration of therapy, thecondition of the subject to be treated, the severity of tinnitus and theefficacy of the particular compound used, age, body weight, generalstate of health, sex, diet, time and route of administration, rate ofexcretion and drug combination ultimately will be decided by theattending physician. The duration of therapy may range between about onehour and several days, weeks or months, and may extend up to chronictreatment. The therapeutically effective amount of the compound to bedelivered may range between about 0.1 nanogram/hour to about 100micrograms hour. The substances of the invention are normallyadministered analogously to other otically-administered compounds. Forexample, ketamine can be otically administered in an amount to treattinnitus, preferably in dosages of about 10 μg/30 ml to about 10,000μg/30 ml, preferably about 500 μg/30 ml, or about 0.01-2 μg per dosage.By the term “dosage” for topical administration, it is meant the amountof agent administered in a single treatment, e.g., about 0.05-1 μgketamine administered to the ear in two drops. Other anti tinnitusagents mentioned herein can be administered analogously, taking intoaccount the potency of the drug.

A therapeutically effective dose is defined as an amount effective tosuppress or reduce NMDA receptor-mediated aberrant activity of theauditory nerve in a treated individual. A therapeutically effective doseis also the amount effective to suppress or reduce tinnitus in theafflicted individual. As stated above, a therapeutically effective dosemay vary, depending on the choice of specific NMDA antagonist fortreatment and on the method of its administration. For example, a higherdose of an intravenously administered NMDA antagonist would be requiredthan that of the same pharmaceutical composition administered locally tothe round window membrane or oval window of the ear. Additionally, alower dose of an NMDA antagonist would be required wherein the NMDAantagonist of the present invention binds the NMDA receptor with ahigher binding affinity than an NMDA antagonist that binds with a loweraffinity. As a result, NMDA antagonists with higher binding affinitiesfor the NMDA receptor are preferred. As stated above, (S)-Ketamine,which binds with a 3-4-fold higher affinity to the PCP binding site ofthe NMDA receptor than (R)-ketamine (Vollenweider et al., Eur.Neuropsychopharmacol. 7: 26-38 (1997)) is a preferred compound for usein the methods of the present invention. The duration of therapy mayalso vary, depending on the specific form of tinnitus for whichtreatment is desired—acute, subacute, or chronic. As a guide, shorterdurations of therapy are preferred and are sufficient when the tinnitusdoes not recur once therapy has ceased. Longer durations of therapy maybe employed for an individual in which tinnitus persists following shorttherapy.

The findings disclosed herein relating to the treatment or prevention oftinnitus, may allow for the manufacture of a medicament for thetreatment or prevention of tinnitus, particularly induced by cochlearexcitotoxicity. In the manufacture of such a medicament, a compound ofthe class of arylcycloalkylamines, preferably of general formula I ormore preferably the arylcycloalkylamine ketamine represented by formulaII may be used. An NMDA receptor antagonist selected from the groupconsisting of 7-chlorokynurenate, D-APS, MK 801 and gacyclidine may alsobe used. Moreover, it is preferred to use a pharmaceutical compositionaccording to the invention which is formulated for topical application,in particular as a solution, gel or other controlled releaseformulation, an ointment or a cream or by means of an invasive drugdelivery techniques, respectively, to be administered topically via theround window membrane or the oval window membrane to the inner ear ordirectly into the inner ear.

EXEMPLIFICATION Example 1 Methods and Materials

We developed and tested an animal model of tinnitus induced by cochlearexcitotoxicity, which was provoked by acoustic trauma. As tinnitus ingeneral is not directly observable, as cochlear excitotoxicity does notresult in tinnitus in all individuals, and as perceptions of tinnitusmay just disappear a few hours after the excitotoxic incident or lastforever, the definition and implementation of such an animal modelrepresented a substantial challenge. These considerations mean forexample that more animals are required to obtain a sufficient number oftinnitus cases for study and to permit observation of tinnitus overtime. As it is unclear whether a case of tinnitus induced by cochlearexcitotoxicity is to last or not, it is advisable to conduct studies inits early stages.

The experiments were performed in two stages. First, the hearing lossfollowing acute acoustic trauma as well as the incidence of tinnituswere evaluated with no therapeutic compound administered. In the secondstage, the efficacy of three pharmaceutical compounds in suppressingtinnitus was tested: S-(+)-Ketamine, a NMDA receptor antagonist(Sigma-Aldrich), 7-chlorokynurenate (7-CK; Sigma-Aldrich), another NMDAreceptor antagonist, which was previously tested in a model ofsalicylate induced tinnitus (Guitton et al., J. of Neuroscience 23 (9):3944-3952 (2003)) as a reference, and D-JNKI-1, a peptide inhibitor ofc-Jun N-Terminal kinase (Xigen S. A.), which was shown to protectagainst auditory hair cell death and hearing loss due to acoustic trauma(Wang et al., J. of Neuroscience 23 (24): 8596-8607 (2003)).Experimental results from the first stage (i.e. no pharmaceuticalcompound used) served as a control.

Animals

Experiments were performed with Long-Evans rats for their superiorlocomotor capacities compared to other rats. During experiments, animalswere caged individually at a constant temperature with a day/night cycleof 12/12 hours. All behavioral tests were performed in the dark phase,the usual period of animal activity, for every animal individually atabout the same time each day. Outside the experiments, the animalsreceived water and nutrition ad libidum. A total of 60 animals wereused: 30 for the first stage (of which 25 were tested by behavioraltechniques and 5 by electrophysiology), and 30 for the second stage with10 for each pharmaceutical compound tested.

Acute Acoustic Trauma

Acoustic trauma was induced by a continuous pure tone of 6 kHz generatedby a waveform synthesizer (Hewlett-Packard 8904A). The animals wereanesthetized and exposed to 130 dB sound pressure level (SPL) for 20minutes, which was routed through a programmable attenuator andpresented to the ears in free field via a JBL 075 earphone positioned 10cm in front of the animal's head. Sound level was measured using acalibrated Bruel and Kjaer microphone (4314) and a Bruel and Kjaercalibrating amplifier (2606).

Behavioral conditioning and testing Animals were conditioned to achieveactive avoidance (Guitton et al., J. of Neuroscience 23 (9): 3944-3952(2003)). Behavioral testing consisted of the performance of a taskwhenever a sound was produced in a conditioning box with an electricalfloor and a climbing pole. Animal conditioning was achieved in a totalof 10 sessions, each lasting between 15 and 20 minutes, with aconditioning stimulus of a pure tone at 50 dB SPL of 3 seconds durationat a frequency of 10 kHz. The unconditional stimulus consisted of anelectric shock to the feet of the animals (3.7 mA) during maximally 30seconds. Interstimulus intervals were 1 second. The electric shocks werestopped by the investigator once the animal correctly climbed onto thepole. Intervals between trials were at least one minute long.

The score was defined as the animal's performance, measured by thenumber of cases when it climbed correctly onto the pole in response tothe sound. As soon as an animal had reached a score of at least 80% inthree consecutive sessions, it was considered successfully conditionedand employed in the experiments.

Experiments were conducted daily with measurements of both score andfalse positive responses during one session of 10 minutes with 10 trialsin total. False positive responses were climbings onto the pole betweentrials without any acoustic stimulation, i.e. during periods of silence.They can be interpreted as the perception of tinnitus, as the animalsare performing the task of climbing onto the pole as if they werehearing the stimulus (Guitton et al., J. of Neuroscience 23 (9):3944-3952 (2003)). Sound stimuli were randomized, and electricalfootshocks were only delivered if the animals didn't climb onto the polein response to sound.

Electrophysiology

The compound action potential (CAP) of the auditory nerve was measuredby an electrode implanted onto the round window membrane of the animals(With a reference electrode placed in a neck muscle). The referenceelectrode and the round window electrode were soldered to a plug fixedon the skull. 10 tone bursts per second (with a duration of 9 ms and arise/fall cycle of 1 ms) generated by an arbitrary function generator(LeCroy Corp., model 9100R), were applied to the animal's ear in freefiled via a JBL 075 earphone. 10 frequencies were tested (2, 4, 6, 8,10, 12, 16, 20, 26, and 32 kHz) with burst levels from 0 to 100 dB SPLin steps of 5 dB. Auditory nerve responses were amplified (Grass P511K,Astro-Med Inc.), filtered (100 Hz to 3 kHz) and averaged on a PC(Dimension Pentium, Dell). CAP amplitudes were measured peak-to-peakbetween the first negative depression N1 and the subsequent positivewave P1. The CAP threshold was defined as the sound intensity (in dBSPL) needed to elicit a measurable response (greater than 5 μV).

Pharmacology

Animals were anaesthetized with a single-dose i.p. injection of 0.3ml/kg of pentobarbital at 6% (Sanofi) and operated under asepticconditions right after the first behavioral testing (day 0). The twobullae were opened through a posterior auricular surgical procedure(dorsal approach). After exposure of the two cochleas, gelfoam (Gelitatampon, B. Braun Medical AG) impregnated with 2.5 μl artificialperilymph containing the pharmaceutical compounds was placed on the eachof the round windows of the two cochleas. The concentration of all threepharmaceutical compounds used was 50 μM. The bullae were then closedwith dental cement (Unifast Trad, GC Corporation), the woundsdisinfected and sutured. The animals were then exposed to thetraumatizing sound. Behavioral tests were resumed 24 hours after theacoustic trauma (day 1), and repeated daily for a total of 8 days.

Statistics

In each behavioral experiment, comparisons of the relevant parameterswere made according to a two-way (group×time, with repeated measures onthe last factor) analysis of variance (ANOVA) in order to test themeasurement effect (group effect), the time effect and the group×timeinteractions. The ANOVA was followed by post hoc comparisons (Tukeytest), Statistical analysis of CAP measurements were made according to aone-way ANOVA followed by Dunnett test. All results were presented asmean±SEM.

Results Stage 1, No Therapeutic Compound Administered

As expected, the traumatizing sound led to a permanent hearing loss (5animals tested with electrophysiology). As shown in FIG. 1, a permanentthreshold shift of maximally 13 dB±2.0 could be observed at 10 kHz onthe 7^(th) day after the acoustic trauma (which had occurred on day 1).

The acoustic trauma also led to a decrease in score (25 animals testedin the behavioral model). As shown in FIG. 2A, the average score droppedsignificantly from the high initial level of day 0 (i.e. before theacoustic trauma) of 87% t 1.6 to a low of 59%±1.0 on day 1, where theacoustic trauma was provoked (p<0.001). Partial functional recoverycould be observed from day 2 (69%±1.2), leveling off on day 4 at anaverage score of 80%±2.0. Statistical analysis of the results showedthat the observed decreases in score were significant (p<0.05), alsofrom day 2 to day 8 (80%±1.4 on the last day). The reduced ability ofanimals to react correctly to the conditioned sound stimulus isconsistent with the fact that the hearing loss provoked by thetraumatizing sound has significantly reduced their ability to hear soundat the frequency of the acoustic stimulus.

Interestingly, it was also found that the number of false positivesdiffered substantially among the animals tested after the acoustictrauma, as shown in FIG. 2B. One group of animals (designated as group1; n=11) displayed no increase in the number of false positives et alleven after the acoustic trauma (0.18 false positives 0.12 on days 0 and1). The remaining 14 animals however delivered a significant increase offalse positives from 0.34±0.13 on day 0 to 4.28±0.22 on day 1. This riseturned out to be reversible for 6 of them (group 2), with the number offalse positives dropping to normal levels again on day 2 and thereafter.The other 8 animals however (group 3) delivered after the transitoryincrease yet another rise in false positives. The maximum of falsepositives in this second phase was observed on day 5 with 3.87±0.29, andthe effect remained statistically significant through day 8 (2.25±0.25false positives on that last day of observation). In other words: therewas first a reversible increase, which was then followed by a permanentincrease in the number of false positive responses to the soundstimulus. This means that after acoustic trauma some animals wereexperiencing no tinnitus et all (group 1), some only in a transitoryform (group 2), and some first in a transitory form and then permanentlyagain for the rest of the observation period. This outcome correspondsin principle to general observations in humans.

Stage 2, Application of Therapeutic Compounds

In order to test whether the mechanism underlying the generation oftinnitus by acoustic trauma was linked to the loss of cochlear haircells and/or to the induction of excitotoxicity, D-JNKI-1 was appliedlocally to the round window membrane. As shown in FIG. 3A, thepharmaceutical compound could not prevent the decrease of score from day0 (88%±2.5) to day 1 (65%±1.7). However, treatment resulted in rapid,full functional recovery to pretraumatic levels on day 2 (90%±2.6),which persisted subsequently (92%±2.0 on day 8).

While D-JNKI-1 prevented permanent hearing loss after acute acoustictrauma, it had no significant effect on the number of false positivesand thus the prevention of tinnitus. As FIG. 3B shows, the patterns offalse positives are almost identical to the ones observed with thecontrol group (FIG. 2B): while group 1 (n=4) showed no increase et all(0.25±0.25 false positives on both days), the two other groups showedagain a statistically significant increase (p<0.05) in the number offalse positives from day 0 (0.33±0.21) to day 1 (4.66±0.42). As forgroup 2 (n=2), the increase was again a short-term, fully reversibleincrease, while the transitory increase in group 3 (n=4) was againfollowed by a permanent rise in the number of false positives (3.50±0.29false positives on day 4 and 2.25±0.25 on day 8), Overall, these resultssuggest that hair cell loss induced by acoustic trauma does not play asignificant role in the generation of tinnitus, and point to cochlearexcitotoxicity as the mechanism at its base.

Local application of the two NMDA receptor antagonists 7-CK andS-(+)-Ketamine yielded results, which were very similar to each other.As shown in FIGS. 4A and 5A, the average score dropped significantlyfrom day 0 to day 1 and then recovered, however at a slower rate than inuntreated animals. In contrast to untreated animals, the stabilizationof the score occurred in the groups of animals treated with NMDAreceptor antagonists only on day 6 (89%±2.3 and 88%±2.5, for animalstreated with S-(+)-Ketamine and 7-CK, respectively). A possibleexplanation for this difference may be that the (partial) blocking ofNMDA receptors is delaying neosynaptogenesis, where they have aneurotrophic effect, and thus retarding functional recovery.

Administration of the two NMDA antagonists had on the other hand asubstantial impact on the number of false positives (FIGS. 4B and 5B).Unlike the untreated animals or those treated with D-JNKI-1, there couldbe no group observed, where a permanent increase in the number of falsepositive responses occurred after an initial transitory increase. Therewas either no increase in false positives et all (group 1; n=5 and n=4for animals treated with S-(+)Ketamine and 7-CK, respectively), wherefalse positives of 0.22±0.22 were observed on days 0 and 1, or just thereversible increase right after the acoustic trauma (group 2; n=5 andn=6 for animals treated with ketamine and 7-CK, respectively), with thenumber of false positives rising from 0.2±0.2 (S-(+)-Ketamine) and0.33±0.21 (7-CK) on day 0 to 5±0.48 (S-(+)Ketamine) and 4.66±0.42 (7-CK)on day 1. There were thus no observations of the onset of a persistenttinnitus following the incidence of transitory tinnitus. These resultsdemonstrate that the local administration of NMDA receptor antagoniststo the cochlea suppresses persisting tinnitus induced by cochlearexcitotoxicity.

Example 2 Methods and Materials

To evaluate the different mechanisms of salicylate and excitotoxicityinduced tinnitus, comparative morphological analysis of the cochlearsensorineural structures as well as Western blot immunodetectionfollowing the two different types of tinnitus inducing incidents wereperformed.

Morphology

Two groups of 3 Long Evans rats each were either treated twice a daywith an intraperitoneal injection of 350 mg/kg of sodium salicylate for2 days or traumatized as described in [0036]. After decapitation of therats under deep anaesthesia (pentobarbital 50 mg/kg), the cochleas wereremoved from the temporal bone and perfused with a fixative solution of2.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.3.They were then processed either for scanning (SEM) or transmissionelectron (TEM) microscopy. For SEM, the otic capsule was dissected outand the stria vascularis, tectorial, and Reissner's membranes wereremoved. After rinsing in PBS (pH 7.3) the samples were dehydrated in agraded series of ethanol (30-100%), critical point-dried in CO₂, coatedwith gold palladium, and examined using a Hitachi S4000 microscope. ForTEM, the cochleas were postfixed in a 1% aqueous solution of osmiumtetroxide for 2 hours, rinsed in phosphate buffer, dehydrated in agraded series of ethanol (30-100%), and embedded in Epon resin.Transverse ultrathin sections of the organ of Corti were taken from theapical half of the cochlea. The sections, mounted on formvar-coated ormesh grids, were stained with uranyl acetate, and lead citrate andexamined using a Hitachi 7100 microscope.

Immunodetection

Three groups of 3 Long Evans rats each were either treated twice over 24hours with an intraperitoneal injection of 350 mg/kg of sodiumsalicylate or traumatized as described in [0036]. The salicylate doseused is known to induce tinnitus (Guitton et al., J. of Neuroscience 23(9): 3944-3952 (2003)). Another group of 3 animals served as control andwas injected i.p. with a NaCl 0.9% solution of the same volume assalicylate treated animals. Samples were obtained after 24 hours in thesalicylate and control groups and 24 hours respectively 5 days postincident for the acoustic trauma group. As had been shown in Experiment1, transitory tinnitus occurred 24 hours after the trauma, andpersisting tinnitus could be observed from the third day on; thereforeit can be expected that persisting tinnitus is present at day 5. Becausesalicylate cannot induce persisting tinnitus, any treatment andmeasurement beyond 24 hours cannot be expected to yield resultsdifferent from those after 24 hours.

Tissues were harvested in cold PBS and homogenized in sample buffer, andthe lysates were centrifuged to remove detergent-insoluble material andseparated on a 10% SDS-PAGE in Tris/Tricine. After gel electrophoresis,proteins were transferred electrophoretically to nitrocellulosemembranes (PVDF transfer membrane Hybond-P, Amersham Pharmacia Biotech,USA). Blots were first incubated with a primary anti-antibody againstthe NMDA NR1 receptor subunit (1/1000 dilution; rabbit polyclonalantibody, Chemicon international, USA), and with a primary antibodyanti-actin (1/50000 dilution, mouse monoclonal anti-β-actin, Sigma, USA)overnight at 4° C. To verify that the molecular weight of the NR1subunit is identical in both brain and cochlea, immunoblots from thebrain of control animals were performed. Then, incubation at 4° C.during 2 hours was done using antibodies anti-rabbit IgG, biotinylatedspecies-specific whole antibody (1/3500, Amersham Lifescience, USA) andanti-mouse IgG, biotinylated species-specific whole antibody (1/3500,Amersham Lifescience, USA). After 5×10 min washes with TBS-T (Trisbuffer saline tween), incubation at 4° C. during 2 hours was done usingstreptavidin alkaline phosphatase conjugate (1/5000, AmershamLifescience, USA). Protein-antibody complexes were revealed withBCIP/NBT (Sigma, USA). Image scanning of Western blots was thenperformed for semi-quantification of expression levels of NR1 and actinproteins using Biorad Fluor-S software (Quantity one).

Results

As expected, the mechanisms underlying salicylate and excitotoxicityinduced tinnitus imply different pathways and result in differentmorphological and physiological outcomes. As shown in FIG. 6,administration of salicylate leaves the stereocilia of sensory outerhair cells (OHC) and inner hair cells (IHC) intact, while those animalswhich were exposed to acoustic trauma display severe damage to OHCstereociliary bundles as well as disarrayed and in some cases even fusedIHC stereocilia. No ultrastructural abnormalities of the IHC synapticcomplex can be seen in case of the salicylate treatment, whereas intraumatized animals a massive and dramatic swelling of the radialafferent dendrites is evident at the basal pole of the IHC in theaffected frequency range area, confirming that excitotoxicity hasoccurred. Numerous vacuoles in the apical part of the IHC and abnormallyshaped stereocilia are present. High magnification of the basal IHC poleshows again no anomalies for the salicylate treated animal. The afferentnerve endings are normal, and a characteristic presynaptic body isclearly visible. Contrary to this, following trauma, swollen anddisrupted nerve endings appear.

FIG. 7 shows the expression of the NR1 subunit of the cochlear NMDAreceptor following exposure to salicylate or acoustic trauma, determinedby Western Blot immunodetection. The salicylate treatment did not induceany significant modification of NR1 NMDA receptor subunit expression (4%higher than control animals). In contrast, acoustic trauma led to aclear overexpression 5 days after the incident (+50% over controlanimals), which is consistent with the observation of persistingtinnitus. The difference in NMDA NR1 expression shows that tinnitusinduced by acoustic trauma is up-regulating NMDA receptors, whereassalicylate does not. This confirms that salicylate induced tinnitus ismediated by a different pathway, as discussed above, FIG. 7 showsfurther that 24 hours post trauma, there is no overexpression detectable(+8%), which suggests that the mechanisms of transitory and permanenttinnitus after acoustic trauma are fundamentally different.

Taken together, the results of the morphological and immunodetectionanalysis confirm the suggested fundamental difference in mechanisms ofaction between salicylate and excitotoxicity induced tinnitus.Excitotoxicity does, unlike salicylate, damage the inner hair cellsynaptic complex and induce an upregulation of NMDA receptors, leadingin turn to the occurrence of persisting tinnitus. As two differentpathways in the regulation of cochlear NMDA responses are involved,efficacy of NMDA receptor antagonists in suppressing persisting tinnitusinduced by cochlear excitotoxicity could realistically not be presumedby a model of salicylate induced tinnitus.

Example 3 Methods and Materials

In order to confirm and extend the results obtained in the previousexperiments, we developed and tested another animal model of tinnitusinduced by cochlear excitotoxicity, which allowed direct measurement ofthe presence of tinnitus, i.e. without using a behavioural model, Martinet al., Proceedings of the Fifth International Tinnitus Seminar,American Tinnitus Association: 127-134 (1996) described a narrowspectral peak at approximately 200 Hz in the spectrum of the ensemblespontaneous activity (“ESA”) of the cochlear nerve in persons sufferingfrom tinnitus. These results were consistent with findings in variousanimal models of from tinnitus. These results were consistent withfindings in various animal models of tinnitus, suggesting that this peakwas indeed an objective correlate of tinnitus. There has been, however,no specific objective evaluation of tinnitus induced by cochlearexcitotoxicity, neither in human beings, nor in animals.

We developed therefore an animal model with guinea pigs that wereexposed to intensive noise, producing acute acoustic trauma, and whoseESA was recorded to control for the absence or presence of tinnitus(“ESA noise trauma model”). The possibility to record tinnitus byelectrophysiological tests provides an excellent tool for evaluating thetherapeutic benefits of pharmaceutical compounds in suppressingtinnitus. The development of the ESA noise trauma model requiredextensive experimentation with different noise trauma protocols as wellas the development and testing of specific measurement tools forextended observation periods in vivo.

The experiment was performed in two stages. First, guinea pigs wereexposed to intense noise stimulation in order to provoke acute acoustictrauma (day 0). Auditory thresholds and ESA were determined and thenfollowed further by repeated electrophysiological measurements. Ifpersisting tinnitus could still be detected after at least 5 days, anNMDA receptor antagonists (ketamine or 7-CK) was applied in a hyaluronicacid formulation onto the round window membrane of the cochlea. Thewaiting period was observed to ensure that no cases of transitory,short-term tinnitus were included; in addition, we were seeking toextend the observation period used in Example 1. After treatment withthe NMDA receptor antagonists, electrophysiological measurements ofhearing thresholds and ESA continued for at least 10 days. The primaryendpoints of the study were to test whether the pharmacologicaltreatment could suppress tinnitus even after its onset, and whether suchtreatment effect was persisting or not. As a control, contralateral earswere exposed to the same noise exposure and followed byelectrophysiology.

Animals

Experiments were performed on adult pigmented guinea pigs (250 to 300grams at study begin). During experiments, animals were cagedindividually. Outside the experiments, the animals received water andnutrition ad libidum. A total of 8 animals were tested in stage 1, ofwhich 3 animals showed persisting and stable tinnitus in their right earfor at least 10 days (minimum 10 days, maximum 30 days), as measured byESA, and were subsequently treated by one of the two NMDA receptorantagonists. In two contralateral left ears, tinnitus was observed, sothat they could be used as a control group.

Acoustic Trauma

Acoustic trauma was induced by a continuous pure tone of 6 kHz generatedby a waveform synthesizer (Hewlett-Packard 8904A). The animals wereanesthetized and exposed to 130 dB sound pressure level (SPL) for 15 to40 minutes, which was routed through a programmable attenuator andpresented to the ears in free field via a JBL 075 earphone positioned 10cm laterally to the animal's ear. Sound level was measured using acalibrated Bruel and Kjaer microphone (4314) and a Bruel and Kjaercalibrating amplifier (2606).

Electrophysiology

A teflon-coated platinum electrode implanted onto the round windowmembranes of both cochleas (with reference electrodes placed in a neckmuscle) measured the ESA and the compound action potential of theauditory nerve (CAP). The surgery was performed using a posteriorauricular surgical procedure (dorsal approach) prior to noise exposure.The reference electrode and the round window electrode were soldered toa plug fixed on the skull using dental cement (Unifast Trad, GCCorporation).

The ESA was recorded from the platinum wire placed in the round windowniche. The signal was amplified by a DC powered amplifier (Radio SpareVIP 20) and A to D converted (48 kHz sampling frequency) by a 24 bitsconverter (National instrument 4474, USA), Spectral analysis of thesignal was performed with the customized computer software LabVIEW 7.1(200 averages, Hanning window, 0-3125 kHz) and the power spectrum wasdisplayed as a function of frequency on a PC computer (Dell Dimension).

The CAP was measured concomitantly with ESA measurements using the sameelectrode. Tone bursts (with a duration of 9 ms and a rise/fall cycle of1 ms, 10 per second) generated by an arbitrary function generator(LeCroy Corp., model 9100R) were applied to the animal's ear in freefiled via a JBL 075 earphone. CAP audiograms were obtained by varyingburst levels from 0 to 100 dB SPL In steps of 5 dB at the frequencies 2,4, 6, 8, 10, 12, 16, 20, and 26 kHz. Auditory nerve responses wereamplified (Tektronic TM 503; gain 2000), filtered (3.5 kHz low pass) andaveraged on a PC (Dimension Pentium, Dell). CAP amplitudes were measuredpeak-to-peak between the first negative depression N1 and the subsequentpositive wave P1. The CAP threshold was defined as the sound intensity(in dB SPL) needed to elicit a measurable response (greater than 5 μV).

Protocol

The ESA was measured just before acute acoustic trauma and 20 minutesfollowing noise exposure on day 0, and then repeatedly for at least 10days as described above. Upon the last ESA measurement confirming thepresence of persisting and stable tinnitus, the animals received theNMDA receptor antagonist treatment onto the round window membrane of thecochlea (treatment day T). Subsequently, ESA was measured again on thefirst day after treatment (T+1) as well as 10 days after treatment(TOO).

Pharmacology

On treatment day (T), the animals were anaesthetized with a single-doses.c. (subcutaneous) injection of Zoletil 50 (Virbac, France)-Rompun 2%(Bayer, Germany) at 55 μl/100 grams and operated under asepticconditions. The right bullae were re-opened through a posteriorauricular surgical procedure (lateral dorsal approach) with particularcare being applied in order not to displace the recording electrode.After exposure of the cochlea, 100 microliters of a gel formulation(0.7% hyaluronic acid, Hylumed Sterile, Genzyme Corp., with phosphatebuffer), with either S-(+)-Ketamine (Sigma-Aldrich) or 7-CK(Sigma-Aldrich) at a concentration of 50 microM were deposited in thebulla. The bulla was then closed with dental cement (Unifast Trad, GCCorporation), the wounds disinfected and sutured. 2 animals receivedKetamine and 1 animal received 7-CK.

Results

Experimentation with various durations showed that noise exposure during40 minutes produced stable and persisting tinnitus. All treated animalswere subjected therefore to a protocol with 130 dB of noise at 6 kHz for40 minutes. This intensive noise exposure resulted in substantialtemporary threshold shifts and permanent hearing loss (mean permanentthreshold shift) of at least 40 dB.

As shown in FIG. 8, the regular spectral peak of ESA centered at 900 Hzto 1 kHz was present prior to the noise exposure. Immediately followingthe noise trauma, the ESA recording showed the emergence of abnormalactivity of the cochlear nerve with a spectral peak centered at 200 to250 Hz, which persisted and remained stable thereafter. Contrary tothis, the regular spectral peak centered at 900 Hz to 1 kHz, whichrepresents the ensemble spontaneous activity of the auditory nerve, wassignificantly reduced and recovered only within 5 days. This evolutionis consistent with the occurrence of temporary threshold shift and thefollowing recovery of auditory function.

On the day of administration of the NMDA receptor antagonist, allanimals were tested to verify the presence of the spectral peak centeredat 200 to 250 Hz in both of their ears. One ear could no longer betested, as the recording electrode no longer functioned properly. Theregular spectral peak centered at 900 Hz to 1 kHz had fully recovered bynow, and hearing threshold levels had recovered partially. Animals 1 and2 received Ketamine and animal 3 received 7-CK, Contralateral earreadings were possible in animals 1 and 2. In all three animals, the ESApeak centered at 200 to 250 Hz completely disappeared on day T1 andremained absent on the follow-up measurement on T10. In thecontralateral ears, the peak remained unaffected on T1 and was stillwell present on T10. Importantly, the hearing loss (permanent thresholdshift) observed on day T remained unaffected by the pharmacologicaltreatment of tinnitus. The results shown in FIG. 8 are fullyrepresentative of the results obtained with the other animals, i.e. thevery same observations were made.

The present results confirm those obtained in the previous experimentsand show that the local administration of NMDA receptor antagonists isvery effective in suppressing tinnitus induced by cochlearexcitotoxicity. It is important to note that the conditions of theexperiment are very close to real life conditions with regard to thepatterns of hearing loss following acute acoustic trauma and thepossibility to treat tinnitus only after its onset. The present resultsclearly show for the first time ever that the tested pharmaceuticalcompounds are not only effective when administered preventively, i.e.before the onset of tinnitus, but also afterwards, and that thetherapeutic window is not limited to just a few hours or days. Thisfinding is very important in view of frequent failures of NMDA receptorantagonists in clinical trials for the treatment of stroke, which hadpreviously shown excellent efficacy in animals when applied prior to theincident or shortly thereafter (see e.g. Stroke Therapy Academicindustry Roundtable, Stroke (30): 2752-2758 (1999)).

1. A method of reducing the perception of tinnitus in a human in needthereof comprising administering to the human a pharmaceuticalcomposition comprising ketamine.
 2. The method of claim 1, wherein thetinnitus is acute.
 3. The method of claim 1, wherein the tinnitusresults from acoustic trauma, presbycusis, ischemia, anoxia, infection,exposure to ototoxic drugs, or sudden deafness.
 4. The method of claim1, wherein the tinnitus results from an occurrence that induces cochlearexcitotoxicity.
 5. The method of claim 1, wherein the tinnitus persistsfor more than 24 hours.
 6. The method of claim 1, wherein thepharmaceutical composition comprises a ketamine analog, a ketaminederivative, a ketamine enantiomer, or pharmaceutically acceptable saltthereof.
 7. The method of claim 6, wherein the ketamine enantiomer is(S)-ketamine or pharmaceutically acceptable salt thereof.
 8. The methodof claim 6, wherein the pharmaceutically acceptable salt ishydrochloride salt.
 9. The method of claim 1, wherein the pharmaceuticalcomposition is a gel.
 10. The method of claim 9, wherein the gel is ahyaluronate gel.
 11. The method of claim 1, wherein the pharmaceuticalcomposition is administered topically via the round window membrane orthe oval window membrane to the inner ear.
 12. The method of claim 1,wherein the pharmaceutical composition is administered by atranstympanic injection.
 13. The method of claim 1, wherein thepharmaceutical composition is delivered to the middle ear.
 14. A methodof ameliorating persistent tinnitus in a human in need thereofcomprising administering to the human a pharmaceutical compositioncomprising ketamine.
 15. The method of claim 14, wherein the tinnitus isacute.
 16. The method of claim 14, wherein the tinnitus results fromacoustic trauma, presbycusis, ischemia, anoxia, infection, exposure toototoxic drugs, or sudden deafness.
 17. The method of claim 14, whereinthe tinnitus results from an occurrence that induces cochlearexcitotoxicity.
 18. The method of claim 14, wherein the tinnituspersists for more than 24 hours.
 19. The method of claim 14, wherein thepharmaceutical composition comprises a ketamine analog, a ketaminederivative, a ketamine enantiomer, or pharmaceutically acceptable saltthereof.
 20. The method of claim 19, wherein the ketamine enantiomer is(S)-ketamine or pharmaceutically acceptable salt thereof.
 21. The methodof claim 19, wherein the pharmaceutically acceptable salt ishydrochloride salt.
 22. The method of claim 14, wherein thepharmaceutical composition is a gel.
 23. The method of claim 22, whereinthe gel is a hyaluronate gel.
 24. The method of claim 14, wherein thepharmaceutical composition is administered topically via the roundwindow membrane or the oval window membrane to the inner ear.
 25. Themethod of claim 14, wherein the pharmaceutical composition isadministered by a transtympanic injection.
 26. The method of claim 14,wherein the pharmaceutical composition is delivered to the middle ear.